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Abstract

Sildenafil citrate (Viagra) is the most widely used drug for treating erectile dysfunction in men. We recently demonstrated that it induces potent protective effects against ischemia-reperfusion (I-R) injury in rabbit hearts through the opening of mitochondrial ATP-dependent K+ channels. In the present study, we investigated the role of the NO-dependent signaling pathway in delayed cardioprotection by sildenafil. Adult male ICR mice were treated with saline or sildenafil (0.7 mg/kg IP) 24 hours before global I-R in the Langendorff mode. Infarct size was reduced from 27.6±3.3% in saline-treated control mice to 6.9±1.2% in sildenafil-treated mice (mean±SEM, P<0.05) without compromising cardiac function. Reverse transcription–polymerase chain reaction revealed a transient increase in endothelial and inducible NO synthase (eNOS and iNOS, respectively) mRNA in sildenafil-treated mice, peaking at 45 minutes (eNOS) and 2 hours (iNOS) after sildenafil injection. The magnitude of mRNA increase was more pronounced for iNOS than for eNOS. In addition, a significant increase in both iNOS and eNOS protein was detected 24 hours after sildenafil treatment. A selective inhibitor of iNOS, 1400W (10 mg/kg IP given 30 minutes before I-R), abolished sildenafil-induced protection (23.7±2.8%, P<0.05 versus sildenafil). These data suggest that the induction of NO synthase isoforms is an essential component of the signaling mechanism for sildenafil-induced delayed preconditioning. However, iNOS appears to be the primary isoform that mediates the robust cardioprotection.

Sildenafil citrate (Viagra) is a selective inhibitor of phosphodiesterase-5, which catalyzes the breakdown of cGMP, one of the primary factors involved in smooth muscle relaxation. It enhances NO-driven cGMP accumulation, which, in turn, causes vasodilatation in the corpus cavernosum. Sildenafil has become the most widely used drug for treating erectile dysfunction in men since its market debut in 1998.1 Interestingly, we recently discovered a powerful preconditioning-like effect of sildenafil in rabbit hearts.2 Both intravenous and oral administration of sildenafil caused significant reduction of infarct size after ischemia-reperfusion (I-R). The protection was abolished by 5-hydroxydecanoate, a selective blocker of mitochondrial ATP-dependent K+ (mitoKATP) channels.2 However, the mechanism by which sildenafil triggers the signaling cascade leading to the opening of mitoKATP remains speculative.2 There is mounting evidence suggesting a role of NO in modulating mitoKATP.3–6 The synthesis of NO is catalyzed by 3 isoforms of NO synthase (NOS), namely, neuronal NOS, inducible NOS (iNOS), and endothelial NOS (eNOS), among which iNOS has been identified as the essential mediator of delayed preconditioning induced by divergent pathophysiological stimuli or pharmacological agents, such as brief episodes of I-R,7 endotoxin derivatives,8,9 G-protein–coupled membrane receptor agonists,10,11 whole-body hyperthermia,12 and systemic hypoxia.13 However, no studies are available showing any link between sildenafil and the activation of a NOS-dependent signaling cascade. The goal of the present report was to determine whether (1) sildenafil induces the synthesis of NOS isoforms in the heart and (2) iNOS mediates the delayed preconditioning effect in the mouse heart.

Materials and Methods

Physiological Studies

Adult male outbred ICR mice were supplied by Harlan Inc (Indianapolis, Ind). Animal experimental protocols were approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University. Viagra pills (Pfizer Inc) were ground into powder and dissolved in saline. The drug solution was filtered (0.45-μm pore size) before intraperitoneal injection. 1400W, an iNOS inhibitor, was obtained from Alexis. We used an isolated perfused mouse heart model subjected to 20 minutes of global ischemia and 30 minutes of reperfusion (Langendorff mode). Myocardial I-R injury was assessed by measuring infarct size, contractile function, and coronary flow as described previously.9

Measurement of NOS Isoforms

Mice were treated with sildenafil (0.7 mg/kg IP), and their hearts were removed at 15, 30, and 45 minutes and 1, 2, 3, 4, and 24 hours after injection (n=3 per group). Three nontreated hearts were used as controls. Tissue samples were ground under liquid nitrogen and homogenized with TRI Reagent (Molecular Research Center) for extracting total RNA, which was reverse-transcribed into cDNA at 50°C for 30 minutes using a OneStep reverse transcription (RT)–polymerase chain reaction (PCR) kit from Qiagen. The oligonucleotide primers were synthesized on the basis of published sequences for murine iNOS, eNOS, and GAPDH15 (Integrated DNA Technology). The RT-PCR products were electrophoresed on 1.5% Tris-acetate-EDTA agarose gel. The target bands were identified on the basis of their specific size using DNA standards.

iNOS and eNOS proteins were measured by Western blots as described previously.13 In brief, triplicate heart samples were collected 24 hours after saline or sildenafil injection and homogenized in ice-cold RIPA buffer (Upstate Biotechnology). The homogenate was centrifuged at 10 000g for 10 minutes at 4°C, and supernatant was recovered as the total cellular protein. Total protein (60 μg) from each sample was separated by SDS-PAGE on 10% acrylamide gels, transferred to a polyvinylidine difluoride membrane, and then blocked with 5% nonfat dry milk in Tris-buffered saline. The membrane was subsequently incubated with a rabbit polyclonal antibody (dilution 1:500, Santa Cruz) reacting specifically to iNOS, eNOS, or actin. The secondary antibody was a horseradish peroxidase–conjugated anti-rabbit IgG (1:1000 dilution, Amersham). The membranes were developed using enhanced chemiluminescence and exposed to x-ray film. The mRNA and protein expression were quantified by scanning each of the RT-PCR or Western blot band using a densitometer (Bioquant 98).

Data Analysis and Statistics

Data are presented as mean±SEM. The difference among the treatment groups or the time points after sildenafil injection was compared by unpaired t test or 1-way ANOVA, followed by the Student-Newman-Keuls post hoc test. A value of P<0.05 was considered significant.

Results and Discussion

Pretreatment of the mice with sildenafil reduced their infarct sizes 24 hours later (6.9±1.2%) compared with saline (control) treatment (27.6±3.3%, P<0.05; Figure 1). The infarct-limiting effect of sildenafil was not associated with compromised ventricular contractile function, ie, stunning (Figure 1, bottom), which is in agreement with the results of Przyklenk and Kloner.16 Sildenafil did not alter preischemic or postischemic coronary flow (data not shown), indicating that its cardioprotective effect may be independent of its vascular response 24 hours later. These results confirmed our previous findings showing a powerful cardioprotective effect of sildenafil in the rabbit heart.2 There was an increase in iNOS and eNOS mRNA and protein expression (Figures 2 and 3). The levels of these transcripts increased transiently, peaking at 45 minutes (eNOS) and 2 hours (iNOS) after sildenafil treatment and returning to baseline levels several hours later (Figure 2). The magnitude of increase was much higher for iNOS compared with eNOS. In addition, sildenafil-induced protection was abolished by the selective iNOS inhibitor 1400W (infarct size 23.7±2.8%, P<0.05 versus sildenafil). 1400W had no significant effect on infarct size compared with the effect of saline (24.5±1.0%, P>0.05 versus saline).

Figure 2. Time course of iNOS and eNOS mRNA expression determined with RT-PCR after sildenafil treatment. Top panel shows representative blots of 3 independent experiments. Bottom panel shows densitometric results averaged from 3 individual hearts for each time point, which were normalized against the GAPDH level for each sample.

Figure 3. Cardiac expression of iNOS and eNOS protein 24 hours after sildenafil treatment. Bar graph shows densitometric quantification averaged from 3 individual hearts for each group, which is normalized against the actin level for each sample.

Several studies have shown that NO derived from iNOS plays a major role in the delayed cardioprotection induced by endotoxin derivatives,8,9 agonists of adenosine or adrenergic receptors,10,11 p38 activator,17 and diazoxide, a mitoKATP opener.6 The role of sildenafil in stimulating the release of NO in the heart is unknown. We provide the first evidence indicating that sildenafil is a potent inducer of iNOS mRNA and protein, which lead to delayed cardioprotection. Although eNOS mRNA and protein also increased, their quantitative expression was lower than that of iNOS. The role of eNOS is not clear in the present study. The complete blockade of cardioprotection with 1400W given before ischemia rules out the role of eNOS in the mediator phase of delayed protection. However, eNOS may play a role in the trigger phase, ie, at the time of sildenafil treatment when NO from eNOS may initiate the signaling cascade leading to iNOS expression, as proposed by Bolli.18,19 The iNOS-catalyzed NO generation could potentially activate guanylate cyclase, resulting in an enhanced formation of cGMP. cGMP may activate protein kinase G, which can subsequently open mitoKATP channels, resulting in the cardioprotective effects as recently reported.20

In conclusion, our results show, for the first time, that sildenafil induces delayed preconditioning, which is primarily mediated by NO derived from iNOS. Further studies are needed to understand the signaling mechanism(s) that leads to the transcription and expression of eNOS and iNOS in the heart. The present study in a model of global I-R further expands our knowledge regarding the cardioprotective effect of sildenafil, which may potentially be used in the treatment of patients with ischemic heart diseases.

Acknowledgments

This study was funded by grants from the NIH (HL-51045, HL-59469 to Dr Kukreja) and the American Heart Association (0060289U to Dr Xi). Dr Yin was supported by an NIH postdoctoral training grant (HL-07537).